RSC96 Cells Research Articles

Ferulic acid alleviates sciatica by inhibiting neuroinflammation and promoting nerve repair via the TLR4/NF-κB pathway.

Sciatica causes intense pain. No satisfactory therapeutic drugs exist to treat sciatica. This study aimed to probe the potential mechanism of ferulic acid in sciatica treatment. Thirty-two SD rats were randomly divided into 4 groups: sham operation, chronic constriction injury (CCI), mecobalamin, and ferulic acid. We conducted RNA sequencing, behavioral tests, ELISA, PCR, western blotting, and immunofluorescence analysis. TAK-242 and JSH23 were administered to RSC96 and GMI-R1 cells to explore whether ferulic acid can inhibit apoptosis and alleviate inflammation. RNA sequencing showed that TLR4/NF-κB pathway is involved in the mechanism of sciatica. CCI induced cold and mechanical hyperalgesia; destroyed the sciatic nerve structure; increased IL-1β, IL-6, TNF-α, IL-8, and TGF-β protein levels and IL-1β, IL-6, TNF-α, TGF-β, TLR4, and IBA-1 mRNA levels; and decreased IL-10 and INF-γ protein levels and IL-4 mRNA levels. Immunohistochemistry showed that IBA-1, CD32, IL-1β, iNOS, nNOS, COX2, and TLR4 expression was increased while S100β and Arg-1 decreased. CCI increased TLR4, IBA-1, IL-1β, iNOS, Myd88, p-NF-κB, and p-p38MAPK protein levels. Treatment with mecobalamin and ferulic acid reversed these trends. Lipopolysaccharide (LPS) induced RSC96 cell apoptosis by reducing Bcl-2 and Bcl-xl protein and mRNA levels and increasing Bax and Bad mRNA and IL-1β, TLR4, Myd88, p-NF-κB, and p-p38MAPK protein levels, while ferulic acid inhibited cell apoptosis by decreasing IL-1β, TLR4, Myd88, p-NF-κB, and p-p38MAPK levels and increasing Bcl-2 and Bcl-xl levels. In GMI-R1 cells, Ferulic acid attenuated LPS-induced M1 polarization by decreasing the M1 polarization markers IL-1β, IL-6, iNOS, and CD32 and increasing the M2 polarization markers CD206, IL-4, IL-10 and Arg-1. After LPS treatment, IL-1β, iNOS, TLR4, Myd88, p-p38MAPK, and p-NF-κB levels were obviously increased, and Arg-1 expression was reduced, while ferulic acid reversed these changes. Ferulic acid can promote injured sciatic nerve repair by reducing neuronal cell apoptosis and inflammatory infiltration though the TLR4/NF-κB pathway.

Enhancing the Effect of Placental Extract on the Regeneration of Crush Injured Facial Nerve.

There is a scarcity of experimental studies on peripheral nerve regeneration using placental extract (PE). This study aimed to investigate the effects of topical PE application on recovery after crush injury to the rat facial nerve using functional, electrophysiological, and morphological evaluations. The viability of the RSC96 Schwann cells treated with PE (0.5~4 mg/ml) increased significantly. Immunoblot test revealed that PE application enhanced the migration of RSC96 cells. Quantitative polymerase chain reaction demonstrated that PE increased the expression of neurotropic genes. The recovery from vibrissa fibrillation in the PE-treated group was superior to that in the control group. The threshold of action potential was also significantly lower in the PE group. Histopathological examination showed that crushed facial nerves treated with PE exhibited larger axons. The surrounding myelin sheaths were more distinct and thicker in the PE-treated group. Hence, PE may be considered a topical therapeutic agent for treating traumatic facial nerve paralysis.

The m6A reader YTHDC2 promotes SIRT3 expression by reducing the stabilization of KDM5B to improve mitochondrial metabolic reprogramming in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a common diabetic complication. Aberrant mitochondrial function causes neurodegeneration under hyperglycemia-induced metabolic stress, which in turn results in DPN progression. m6A and m6A reader (YTHDC2) are closely related to diabetes and diabetes complications, while the role of YTHDC2 in regulating mitochondrial metabolism in DPN needs to be further probed. For HG treatment, Schwann cells (RSC96) were subjected to D-glucose for 72h. db/db mice were used as the diabetic mouse model. Me-RIP assay was performed to evaluate KDM5B m6A level. RNA degradation assay was conducted to examine KDM5B mRNA stability. In addition, OCR and ECAR were examined by XF96 Analyzer. Moreover, the content of ATP and PDH activity in RSC96 cells were detected using kits, and the level of ROS was detected using MitoSOX staining. RIP, RNA pull-down and dual-luciferase reporter gene assays were carried out to verify the binding relationships between YTHDC2, KDM5B and SIRT3. We first observed that KDM5B expression and KDM5B mRNA stabilization were significantly increased in DPN. The m6A reader YTHDC2 was lowly expressed in DPN. Meanwhile, YTHDC2 over expression decreased KDM5B mRNA stability in an m6A-dependent manner. Our results also revealed that YTHDC2 overexpression resulted in reduced ROS level and increased ATP level, PDH activity, OCR and ECAR in HG-treated Schwann cells, while these effects were reversed by KDM5B overexpression. Additionally, SIRT3 served as the target of YTHDC2/KDM5B axis in regulating mitochondrial metabolism in DPN. Taken together, YTHDC2 promoted SIRT3 expression by reducing the stabilization of KDM5B to improve mitochondrial metabolic reprogramming in DPN.

An Eco-Friendly Antheraea Pernyi Silk Gland Protein/Sodium Alginate Multiple Network Hydrogel as Potential Drug Release Systems

To improve the versatility of the sodium alginate-loaded bio-hydrogels, Antheraea pernyi silk gland protein/sodium alginate drug-loaded hydrogels were prepared by using an eco-friendly multiple network cross-link technology. Fourier transform infrared (FT-IR) spectroscopy and UV-Vis spectrophotometer were used separately to evaluate the chemical structure and drug release behavior of drug-loaded hydrogels. The antibacterial drug carrier gels were evaluated by using inhibition zone test against the S. aureus and E. coli. The CCK-8 assay was used to assess the biocompatibility of drug loaded hydrogels. The FT-IR results showed that there was a strong interaction within the drug loaded hydrogels, and the ASGP was beneficial to enhance the interaction within the drug loaded hydrogels. UV-Vis spectrophotometer results indicated the cumulative release reached 80% within 400 min. Antibacterial bio-hydrogels had a good antibacterial property, especially the antibacterial bio-hydrogels with bacitracin exhibits superior to other antibacterial agents. The drug-loaded bio-hydrogels exhibited the adhesion and proliferation of RSC96 cells and perfected biocompatibility. This provides a new idea for further research and development of tissue-friendly drug-loaded biomaterials.

Identification of pyroptosis‑related genes in neuropathic pain based on bioinformatics analysis.

Pyroptosis is defined as inflammation-induced programmed cell death. However, gene expression levels related to pyroptosis and their role in neuropathic pain (NP) remain unclear. The present study aimed to develop and validate an NP-predictive signature based on the genes associated with pyroptosis. Gene expression level profiles were downloaded from the Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the pyroptotic genes most highly associated with NP. NP-related pyroptosis gene signature was constructed using multivariate logistic regression. A rat model of neuropathic pain was established through chronic constriction injury to analyse the inflammatory infiltration and myelin damage around the sciatic nerve, and examine the expression levels of macrophage markers S100 calcium-binding protein β (S100β) and ionized calcium-binding adapter molecule 1 (Iba-1). Finally, flow cytometry analysis was used to examine the lipopolysaccharide (LPS)-induced cell death ratio of RSC96 cells (Schwann cells), while the expression levels of LPS-induced pyroptosis-related genes in RSC96 cells were measured via reverse transcription-quantitative PCR. The results demonstrated that pyroptosis-related genes (gasdermin D, NLR family pyrin domain containing 3, neuronal apoptosis inhibitory protein and NLR family CARD domain containing 4) were identified to increase the risk of NP. NP-related pyroptosis signatures were constructed based on these four genes. Moreover, the high-risk group had a higher level of macrophage infiltration compared with the low-risk group, as determined by the CIBERSORT algorithm. H&E staining results showed that the myelin structure of the sciatic nerve tissue of chronic constriction injury (CCI) rats was destroyed and inflammatory cells infiltrated around neurons. The results of immunohistochemistry showed that compared with in the sham group, the expression levels of Iba-1 and sS100β in the sciatic nerve of the CCI group were increased. Furthermore, the expression levels of cell death and pyroptosis-related genes in Schwann cells induced by LPS were increased compared with in the control group. In conclusion, an NP-related pyroptosis gene signature was constructed based on four pyroptosis-related genes and it was found that the expression of pyroptosis-related genes was upregulated in the early steps of the neuroinflammatory process in RSC96 cells.

Ginkgetin promotes proliferation and migration of Schwann cells via PIGF/p38 MAPK signaling pathway

The proliferation and migration of Schwann cells is pivotal to peripheral nerve injury (PNI) repair. Recent studies have revealed that Ginkgetin has neuroprotective effects. Hence, we focused on identifying whether Ginkgetin could regulate the proliferation and migration of Schwann cells, thereby contributing to the repair of PNI. Rat Schwann cells RSC96 were treated with different concentrations of Ginkgetin. Short hairpin RNA targeting phosphatidylinositol glycan anchor biosynthesis class F (shPIGF) was employed to investigate the effects of PIGF on Ginkgetin-induced RSC96 cells. Viability of RSC96 cells was estimated via cell counting kit-8 (CCK-8) assay and proliferation of the cells was assessed by 5-ethynyl-2′-deoxyuridine (EdU) assay. Migration was estimated via wound healing assay and invasion was evaluated through transwell assay. Western blot was employed to test the expressions of PIGF, protein-38 (p38), and phosphorylated p-38 (p-p38). Ginkgetin (50 or 100 μg/ml) increased the viability, proliferation, migration, and invasion of RSC96 cells, up-regulated PIGF expression and raised the ratio of p-p38/p38, which were all reversed by PIGF silencing. Ginkgetin promotes proliferation, migration, and invasion of Schwann cells via PIGF/p38 MAPK signaling pathway.

Engineering Three-Dimensional Bendable Helix Conduits for Peripheral Nerve Regeneration via Hybrid Electrotechnologies

Nerve guidance conduits (NGCs) are considered as the best alternative approach to autologous nerve grafting for treating peripheral nerve injuries (PNI). The bendable NGCs are desirable in practical use but are still difficult to fabricate. In this study, the hybrid electrotechnologies combining electrohydrodynamic (EHD) printing and electrospinning were applied to produce a novel bendable helix conduit with tunable flexibility. Helical polycaprolactone (PCL) foundations were constructed via EHD printing for mechanical support, on which electrospinning PCL membrane was coated to form the bendable helix conduits and keep the entire structure stable. The effects of helical foundation with different helical height on the properties of NGCs were analyzed. In addition, these three-dimensional (3D) bendable helix conduits had excellent resistance to compression and fatigue. Moreover, the bendable conduits had higher specific surface area, which is beneficial to the adsorption of RSC96 cells. Therefore, the 3D bendable helix conduits show great potential in clinical application for treating PNI.

CdTe quantum dots trigger oxidative stress and endoplasmic reticulum stress-induced apoptosis and autophagy in rat Schwann cell line RSC96.

In the current study, the cytotoxicity and mechanisms of cadmium telluride quantum dots (CdTe QDs) on RSC96 cells were evaluated by exposing different doses of CdTe QDs for 24 h. Two types of cell death, including apoptosis and autophagy, as well as two important organelles, mitochondria and endoplasmic reticulum, were focused after CdTe QDs exposure. The results showed that CdTe QDs induced apoptosis in RSC96 cells in a concentration-dependent manner; promoted the accumulation of intracellular reactive oxygen species; decreased the mitochondrial membrane potential; caused the release of cytochrome c; and also increased the expression of Bcl-2 associated X protein, caspase-3, and cytochrome c proteins and decreased the expression of Bcl-2 protein. Further results also confirmed that CdTe QDs could be internalized by RSC96 cells, and the exposure and internalization of CdTe QDs could induce excessive endoplasmic reticulum stress in the cells, and the expression levels of binding immunoglobulin protein, C/EBP homologous protein, and caspase12 proteins were increased in a concentration-dependent manner. Moreover, autophagy-related proteins LC3II, Beclin1, and P62 all increased after CdTe QDs exposure, suggesting that CdTe QDs exposure both promoted autophagosome formation and inhibited autophagosome degradation, and that CdTe QDs affected the autophagic flow in RSC96 cells. In conclusion, CdTe QDs are able to cause apoptosis and autophagy in RSC96 cells through mitochondrial and endoplasmic reticulum stress pathways, and the possible neurotoxicity of CdTe QDs should be further investigated.

MicroRNA-7a-5p ameliorates diabetic peripheral neuropathy by regulating VDAC1/JNK/c-JUN pathway.

The pathogenesis of diabetic peripheral neuropathy (DPN) is complex, and its treatment is extremely challenging. MicroRNA-7a-5p (miR-7a-5p) has been widely reported to alleviate apoptosis and oxidative stress in various diseases. This study aimed to investigate the mechanism of miR-7a-5p in DPN. DPN cell model was constructed with high-glucose-induced RSC96 cells. Cell apoptosis and viability were detected by flow cytometry analysis and cell counting kit-8 (CCK-8) assay respectively. The apoptosis and Jun N-terminal kinase (JNK)/c-JUN signalling pathway-related proteins expression were detected by Western blotting. The intracellular calcium content and oxidative stress levels were detected by flow cytometry and reagent kits. Mitochondrial membrane potential was evaluated by tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) staining. The targeting relationship between miR-7a-5p and voltage-dependent anion-selective channel protein 1 (VDAC1) was determined by RNA pull-down assay and dual-luciferase reporter gene assay. The streptozotocin (STZ) rat model was constructed to simulate DPN in vivo. The paw withdrawal mechanical threshold (PTW) was measured by Frey capillary line, and the motor nerve conduction velocity (MNCV) was measured by electromyography. MiR-7a-5p expression was decreased, while VDAC1 expression was increased in HG-induced RSC96 cells and STZ rats. In HG-induced RSC96 cells, miR-7a-5p overexpression promoted cell proliferation, inhibited apoptosis, down-regulated calcium release, improved mitochondrial membrane potential and repressed oxidative stress response. MiR-7a-5p negatively regulated VDAC1 expression. VDAC1 knockdown improved cell proliferation activity, suppressed cell apoptosis and mitochondrial dysfunction by inhibiting JNK/c-JUN pathway activation. MiR-7a-5p overexpression raised PTW, restored MNCV and reduced oxidative stress levels and nerve cell apoptosis in STZ rats. MiR-7a-5p overexpression ameliorated mitochondrial dysfunction and inhibited apoptosis in DPN by regulating VDAC1/JNK/c-JUN pathway.

456-P: HMGB1 Inhibition Protects Schwann Cells from High Glucose–Induced Cytotoxicity

Objective: Damage to Schwann cells play a crucial role in diabetic peripheral neuropathy (DPN) . To observe whether HMGB1 inhibitor (Glycyrrhizic acid, GA) can prevent diabetic Schwann cells damage and its potential mechanisms. Methods: RSC96 cells were divided into 5 groups: NG group (5.6 mmol/L) and HG group (25.0 mmol/L) , HG+GA (1μM) group, HG+GA (10μM) , HG+GA (100μM) . Small interfering RNA was used to knock out HMGB1. CCK8 assay was used to detect the proliferation of RSC96 cells. The levels of inflammatory factors were determined by ELISA. The mRNA expression of NGF and neuritin-1 was detected by qRT-PCR. The expression levels of NSE, cleaved-caspase-3, RAGE, p38MAPK, ERK, JNK and NF-κBp65 in cells were measured by western blot. Results: GA at the concentration of μM for 24h had significant inhibitory effect on HMGB1 synthesis and secretion (P<0.05) . Schwann cell viability in high glucose was restored due to GA and siHMGB1 (P<0.05) . GA intervention and siHMGB1 potently prevented inflammatory substance generation (TNF-α, IL-1β, IL-6, MCP-1 and ICAM-1) , alleviated the neurotrophic factor reduction (NGF and neuritin-1) , and suppressed cellular apoptosis related protein activation (cleaved caspase-3) in Schwann cells exposed to high glucose ambience (P<0.05) . Moreover, we found that RAGE expression, p38MAPK phosphorylation and nuclear NF-κBp65 expression in high glucose-stimulated Schwann cells was reversed by GA or siRNA interference with HMGB1 (P<0.05) . Conclusions: GA and siHMGB1 can not only inhibit HMGB1 expression in Schwann cells exposed to high glucose, but also enhance cell viability. GA and siHMGB1 restored neurotrophic factors level and reduce cleaved caspase-3 expression in Schwann cell due to its anti-inflammatory ability, which may be related to the inhibition of RAGE/p38MAPK/nuclear NF-κBp65 pathway. HMGB1 may implicate in Schwann cells lesion in high glucose milieu and HMGB1 inhibition protects Schwann cells from high glucose induced cytotoxicity. Funding National Natural Science Foundation of China Grant Award (81700723)

Mammalian Sterile 20-Like Kinase 1 Mediates Neuropathic Pain Associated with Its Effects on Regulating Mitophagy in Schwann Cells.

Myelin degradation initiated by Schwann cells (SCs) after nerve injury is connected to the induction and chronicity of neuropathic pain (NP). Mitophagy, a selective clearance of damaged mitochondria via autophagy, contributes to the maintenance of normal function in SCs. Mitochondrial function and mitophagy activity are highly modulated by mammalian ste20-like kinase1 (Mst1). However, whether Mst1 can regulate mitophagy in SCs to play a role in NP remains poorly understood. In the present study, Sprague-Dawley rats were subjected to chronic constriction injury (CCI) on the sciatic nerve to induce NP. Small interfering RNA of Mst1 was applied to the injured sciatic nerve to knockdown Mst1. Behavioral tests were performed to evaluate NP, and myelin degeneration was assessed by transmission electron microscope and immunofluorescence. Autophagy and mitophagy were detected in the injured sciatic nerve and cultured SCs (RSC96 cells) by Western blot. ROS level, mitochondria membrane potential, and apoptosis were assessed in vitro via flow cytometry and Western blot. Mst1 knockdown alleviated mechanical allodynia and thermal hyperalgesia in the CCI-induced NP model and rescued myelin degeneration of the injured nerve. Meanwhile, CCI-increased levels of Parkin and p62 were reversed by Mst1 knockdown. In vitro RSC96 cells were subjected to starvation to induce mitophagy. Protein levels of mitochondrial Parkin and mitochondrial p62 significantly increased after Mst1 knockdown, while those in the cytosol diminished indicate that the translocation of Parkin and p62 from the cytosol to the mitochondria was promoted by the knockdown of Mst1. In addition, Mst1 knockdown reduced ROS level and apoptosis activity, while enhancing mitochondria membrane potential in RSC96 cells. The study showed that Mst1 knockdown alleviated CCI-induced NP, associated with enhanced Parkin recruitment to mitochondria and subsequent mitophagy degradation, thus preserving mitochondrial function and myelin integrity.

Melatonin promotes peripheral nerve repair through Parkin-mediated mitophagy

Schwann cells (SCs) are the major glial cells in peripheral nervous system. They unsheathe and myelinate axons and play an essential role in peripheral nerve regeneration. Several studies report that Parkin-mediated mitophagy is associated with various diseases. Melatonin promotes proliferation of central glial cells. Little is known about the effect of melatonin and Parkin-mediated mitophagy on peripheral nerve repair.In this study, using a rat model of a peripheral nerve injury (PNI) and in vitro model established by RSC96 cells treated with tert-butyl hydroperoxide (TBHP), we found that Parkin-mediated mitophagy can effectively reduce the production of mitochondrial reactive oxygen species (ROS), maintain the balance of mitochondrial membrane potential, maintain autophagic flux, and inhibit mitochondrial apoptosis. At the same time, we found that the increase of Parkin under stress is a manifestation of the RSC96 cells' resistance to oxidative stress to maintain RSC96 cells' balance. In our experiment, melatonin is similar to a Parkin agonist, up-regulating the expression of Parkin, enhancing all the positive results of Parkin in a stress state, such as inhibiting active oxygen production, maintaining autophagic flux, and inhibiting mitochondrial apoptosis. In addition, we design in vivo experiments to verify in In vitro experiments. In in vivo, melatonin promotes the expression of Parkin, maintains autophagic flux, inhibits apoptosis, promotes myelin regeneration, reduces the regeneration of collagen fibers around damaged tissues, and promotes peripheral nerve repair. When adenovirus was used to down-regulate the expression of Parkin, we found that all the positive effects of melatonin were attenuated.Collectively, these findings indicate that melatonin upregulates Parkin-mediated mitophagy and promotes peripheral nerve repair. The results provide a basis for development of effective drugs for PNI treatment.

Development of a novel vector for siRNA delivery based on arginine‐modified polyvinylamine

AbstractAn ideal carrier that delivers small interfering RNA (siRNA) should be designed based on two criteria: high gene transfection efficiency and low cytotoxicity. In this work, arginine was used to modify polyvinylamine (PVAm) to form a novel PVAm derivative (PVAm‐Arg) for siRNA delivery and the derivative was characterized using Fourier transform infrared spectroscopy. PVAm‐Arg/siRNA complexes were formed by the self‐assembly method. The zeta potential and size of the complexes were measured using electrophoretic light scattering and dynamic light scattering, respectively. The siRNA binding capacity of PVAm‐Arg was assessed using gel retardation assay. The results showed that PVAm‐Arg derivatives can bind siRNA effectively and form complexes with sizes of about 72 ± 2 nm. What is more, the ability of PVAm‐Arg to condense siRNA was better than that of PVAm. Cytotoxicity assay with RSC96 cells also showed that PVAm‐Arg had lower cytotoxicity compared with PVAm. In in vitro cellular uptake assay, PVAm‐Arg showed good transfection efficiency. Therefore, we concluded that PVAm‐Arg would be a promising candidate as a gene delivery vector. © 2022 Society of Industrial Chemistry.

Attenuation of Oxidative Stress-Induced Cell Apoptosis and Pyroptosis in RSC96 Cells by Salvianolic Acid B.

To determine whether salvianolic acid B (Sal B) exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis. RSC96 cells were primarily cultured with DMEM (5.6 mmol/L glucose), hyperglycemia (HG, 125 mmol/L glucose) and Sal B (0.1, 1, and 10 µ mol/L). Cells proliferation was measured by 3-(4, 5-cimethylthiazol-2-yl)-2, 5-dilphenyltetrazolium bromide assay. Reactive oxygen species (ROS) generation and apoptosis rate were detected by flow cytometry analysis. Western blot was performed to analyze the expressions of poly ADP-ribose polymerase (PARP), cleaved-caspase 3, cleaved-caspase 9, Bcl-2, Bax, NLRP3, ASC, and interleukin (IL)-1β. Treatment with HG at a concentration of 125 mmol/L attenuated cellular proliferation, while Sal B alleviated this injury (P<0.05). In addition, Sal B inhibited HG-induced ROS production and apoptosis rate (P<0.05). Furthermore, treatment with Sal B down-regulated HG-induced PARP, cleaved-caspase 3, cleaved-caspase 9, Bax, NLRP3, ASC, and IL-1β expression, but mitigated HG-mediated down-regulation of Bcl-2 expression (P<0.05). Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.

DHI Increases the Proliferation and Migration of Schwann Cells Through the PI3K/AKT Pathway and the Expression of CXCL12 and GDNF to Promote Facial Nerve Function Repair.

The facial nerve is one of the vulnerable nerves in otolaryngology. Repair and recovery of facial nerve injury have a high priority in clinical practice. The proliferation and migration of Schwann cells are considered of great significance in the process of nerve injury repair. Danhong injection (DHI), as a common drug for cardiovascular and cerebrovascular diseases, has been fully certified in neuroprotection research, but its role in facial nerve injury is still not clear. Our study found that DHI can promote the proliferation and migration of RSC96 cells, a Schwann cell line, and this effect is related to the activation of the PI3K/AKT pathway. LY294002, an inhibitor of PI3K, inhibits the proliferation and migration of RSC96 cells. Further studies have found that DHI can also promote the expression of CXCL12 and GDNF at gene and protein levels, and CXCL12 is, while GDNF is not, PI3K/AKT pathway-dependent. Animal experiments also confirmed that DHI could promote CXCL12 and GDNF expression and promote facial nerve function recovery and myelin regeneration. In conclusion, our in vitro and in vivo experiments demonstrated that DHI could promote the proliferation and migration of Schwann cells through the PI3K/AKT pathway and increase the expression of CXCL12 and GDNF to promote facial nerve function repair.

The synergistic effect of palmitic acid and glucose on inducing endoplasmic reticulum stress-associated apoptosis in rat Schwann cells.

Diabetic peripheral neuropathy (DPN) is a common long-term complication of diabetes mellitus accompanied with hyperglycemia and hyperlipidemia. Both high blood glucose and high blood lipids are key pathogenies for DPN. This research aims to investigate whether the combination of glucose (Glu) and palmitic acid (PA) played a synergistic role in the pathogenesis of DPN. The proliferation rate of Rat Schwann cell line RSC96 cells stimulated by different concentrations of Glu and PA were analyzed by CCK-8 assay. After the IC50 was detected for each drug, the RSC96 cells were divided into control, Glu, Glu+PA, PA, and BSA groups. The apoptosis of RSC96 cells in different groups were detected by flow cytometry. The effects of Glu and/or PA on endoplasmic reticulum (ER) stress-associated apoptotic signaling pathways were determined by Western blot and qPCR. Both Glu and PA showed similar inhibition on the proliferation of RSC96 cells in a dose-dependent manner. However, PA induced stronger apoptosis of RSC96 cells than glucose and significantly increased the levels of X-box-binding protein-1 (XBP1), C/EBP homologous protein (CHOP), and eIF2α phosphorylation, which are key proteins regulating endoplasmic reticulum (ER) stress-associated apoptotic signaling pathways. The combination of Glu and PA induced the strongest apoptosis in RSC96 cells and also activated ER stress-associated apoptotic signaling pathways. These results verified the synergistic effect of Glu and PA on inducing ER stress-associated apoptosis in RSC96 cells, and PA even induced stronger apoptosis in RSC96 cells than Glu. The present research indicated that hyperglycemia and hyperlipidemia might exert a synergistic damage during the pathogenesis of DPN, suggesting that blood lipid control is as important as blood glucose control for DPN patients.

Glycyrrhizic acid promotes sciatic nerves recovery in type 1 diabetic rats and protects Schwann cells from high glucose-induced cytotoxicity.

The present study aims to investigate the therapeutic effect and mechanism of glycyrrhizic acid (GA) in diabetic peripheral neuropathy (DPN). GA significantly mitigated nerve conduction velocity (NCV) deficit and morphological abnormality and reduced high-mobility group box-1 (HMGB1) expression in the sciatic nerves of diabetic rats independent of blood glucose and body weight. Notably, GA alleviated the increase of HMGB1 and the decrease of cell viability in high glucose-stimulated RSC96 cells. Furthermore, GA obviously reduced the concentration of inflammatory cytokines in the sciatic nerves of diabetic rats and supernatants of high glucose-exposed RSC96 cells, then restored the decreased expression levels of nerve growth factor (NGF) and neuritin-1, and the increased expression levels of cleaved caspase-3 and neuron-specific enolase. Additionally, GA markedly inhibited receptor for advanced glycation end products (RAGE) expression, p38MAPK phosphorylation, and the nuclear translocation of NF-κBp65 in diabetic rats and high glucose-exposed RSC96 cells. The promotional effect of high glucose in RSC96 cells was diminished following Hmgb1 siRNA treatment. Our findings indicate that GA may exert neuroprotection on DPN by suppressing HMGB1, which lead to extenuation of inflammation response, balance of NGF, neuritin-1 and caspase-3, as well as inactivation of RAGE/p38MAPK/NF-κBp65 signaling pathway.

Sustained delivery of vascular endothelial growth factor mediated by bioactive methacrylic anhydride hydrogel accelerates peripheral nerve regeneration after crush injury.

Neurotrophic factors, currently administered orally or by intravenous drip or intramuscular injection, are the main method for the treatment of peripheral nerve crush injury. However, the low effective drug concentration arriving at the injury site results in unsatisfactory outcomes. Therefore, there is an urgent need for a treatment method that can increase the effective drug concentration in the injured area. In this study, we first fabricated a gelatin modified by methacrylic anhydride hydrogel and loaded it with vascular endothelial growth factor that allowed the controlled release of the neurotrophic factor. This modified gelatin exhibited good physical and chemical properties, biocompatibility and supported the adhesion and proliferation of RSC96 cells and human umbilical vein endothelial cells. When injected into the epineurium of crushed nerves, the composite hydrogel in the rat sciatic nerve crush injury model promoted nerve regeneration, functional recovery and vascularization. The results showed that the modified gelatin gave sustained delivery of vascular endothelial growth factors and accelerated the repair of crushed peripheral nerves.

Biocompatible Polyurethane Conduit Grafted with Vascular Endothelial Growth Factor-Loaded Hydrogel Repairs the Peripheral Nerve Defect in Rats.

Artificial nerve guidance conduits (NGCs) can be potentially used to address the problems of peripheral nerve defects. The biomaterial polyurethane (PU) has already been used to construct NGCs. However, the use of a combination of PU-based NGCs and other bioactive cues, such as extracellular matrix proteins and growth factors, has not been reported yet. A PU conduit grafted with a vascular endothelial growth factor (VEGF)-loaded hydrogel (abbreviated as PU/Gel/VEGF conduit) is fabricated. The leachate generated during the use of the PU/Gel/VEGF conduit could facilitate the proliferation, migration, and expression of the neural marker S100β in RSC96 cells (in vitro). The walking track and target muscle are analyzed, and it is observed that PU/Gel/VEGF conduits promote the functional recovery of the injured side. Various histological staining analyses are carried out, and the results reveal that the PU/Gel/VEGF conduit effectively improves the extent of nerve regeneration achieved. The number of blood vessels developed during the regeneration of the axons in the PU/Gel/VEGF group (attributable to the pro-angiogenic effect of the functional NGC) is higher than the number of blood vessels developed in the PU/Gel conduit. Overall, the results indicate that PU/Gel/VEGF conduits could promote the process of peripheral nerve regeneration.

Lycorine improves peripheral nerve function by promoting Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation in diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus and no effective therapy is approved. Here, lycorine, a natural alkaloid, was identified as a potential drug for DPN by the bioinformatics analysis of GEO datasets and Connectivity Map database. Lycorine administration improved peripheral nerve function and autophagy-associated proteins of diabetic mice. Again, in vitro high glucose-cultured rat Schwann cells (RSC96) showed enhanced autophagosome marker LC3-II with the treatment of lycorine. Additionally, beclin-1 and Atg3 were decreased in high glucose-stimulated RSC96 cells, which were reversed by lycorine treatment. Furthermore, DPN-associated differentially expressed genes (DEGs) from GEO datasets and lycorine-drug targets from PubChem and PharmMapper were visually analyzed and revealed that MMP9 was both DPN-associated DEGs and lycorine-drug target. Functional enrichment analysis of MMP9-relevant genes showed that cell energy metabolism was involved. Moreover, lycorine reduced high glucose-enhanced MMP9 expression in RSC96 cells. Overexpression of MMP9 attenuated lycorine-induced the expression of beclin-1, Atg3 and LC3-II in high glucose-cultured RSC96 cells. In addition, AMPK pathway activation was confirmed in lycorine-treated high glucose-cultured RSC96 cells. Then AMPK pathway inhibition attenuated lycorine-reduced MMP9 expression in high glucose-treated RSC96 cells. Molecular docking analysis revealed that lycorine bound the domain of AMPK containing Thr 172 site, which affected AMPK (Thr 172) phosphorylation. Finally, AMPK pathway activation and MMP9 downregulation were also revealed in the sciatic nerves of diabetic mice administrated with lycorine. Taken together, lycorine was advised to promote Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation-induced LC3-II transformation in diabetic peripheral neuropathy.